Compositions for use in the promotion of intestinal muscle growth and development and associated intestinal motility

ABSTRACT

This invention relates to nutritional compositions comprising at least one N-acetylated oligosaccharide for use in the promotion of the development and/or of the growth of the intestinal muscles, in the promotion of contractile capacity and/or the motility in the intestine, in the promotion of enteral feeding tolerance and in prevention and/or treatment of small intestinal bacterial overgrowth (SIBO) in an infant, a young child or, when the composition is a growing-up milk, in a child.

FIELD OF THE INVENTION

This invention relates to nutritional compositions comprising at leastone N-acetylated oligosaccharide for use in the promotion of thedevelopment and/or of the growth of the intestinal muscles, in thepromotion of contractile capacity and/or the motility in the intestine,in the promotion of enteral feeding tolerance and in prevention and/ortreatment of small intestinal bacterial overgrowth (SIBO) in an infant,a young child or, when the composition is a growing-up milk, in a child.

BACKGROUND OF THE INVENTION

The layer of smooth muscle in the intestine, also referred to asmuscular coat, muscular fibers, muscularis propria or muscularisexterna, is adjacent to the submucosa. The development of these musclesplay a critical role in the development of intestinal function and inparticular in the development of contractility and motility. It istherefore important for the intestine muscle of an infant, a young childor a child to have optimal development and growth. One essential aspectof intestinal muscle growth and development is the increase of musclethickness, contributing to increase in muscle mass. Indeed, too thin amuscle layer would impair the functionality of such muscle and thus themotor function of the intestine.

Gut motility is the motor function of the intestine. It is key to ensuresuccessful digestion, absorption of nutrients and excretion of theun-digested materials, of the bacteria and of the toxic components inthe lumen. Motility is operated by contraction of the smooth muscle allalong the gastro-intestinal tract. Such contractions are effective inthe propulsion of the chyme along the gastrointestinal tract, leading tobowel movements, and in mixing the ingested materials to ensureappropriate digestion.

Peristalsis is the main type of propulsive motility, which takes placein particular in the esophagus and in the small intestine. A ring ofmuscle gets contracted, while the immediate distal portion of thegastrointestinal tract relaxes, thus pushing the bolus towards the anus.

Mixing is operated by segmentation contraction. Alternative relaxationand contraction of the intestinal muscle are effective in chopping andmixing the ingested materials. This mechanism is necessary to ensureproper contact of the whole bolus with digestive enzymes, as well asoptimal absorption of the nutrients.

Abnormal intestinal motility can be due either to an impaired muscularor nervous function in the intestine. Thus the characteristics of thesmooth muscle in the intestine plays an important role in thedevelopment and maintenance of an optimal intestinal motility. Forexample a muscle layer that would be of insufficient thickness couldlead to impaired intestinal motility. Indeed, the contractile capacityof the intestine is proportional to the muscle mass (see for exampleTanovic et al.; Alterations in Intestinal Contractility duringInflammation Are Caused by Both Smooth Muscle Damage and SpecificReceptor-mediated Mechanisms; Croat Med J. 2006 April; 47(2): 318-326).

Impaired motility is associated with adverse effects such as impairedtolerance to enteral feeding and small intestinal bacterial overgrowth.Both of these effects are generated by the insufficient contractibilityof the smooth muscle, leading to insufficient digestion of enteral feedand insufficient ability to excrete bacteria.

The link between impaired motor function in the intestine and impairedtolerance to enteral feeding has been extensively documented in theliterature. For example, Bisset et al.; Postprandial motor response ofthe small intestine to enteral feeds in preterm infants; Archives ofDisease in Childhood, 64 (1989): 1356-1361 report this problem inpreterm infants.

Small intestine bacterial overgrowth (SIBO) is a disturbance of thequantitative and qualitative intestinal microbiota balance and maycontribute to severe health problems. The relationship between alteredsmall intestinal motility has been established in the literature (seefor example Roland et al; Small Intestinal Transit Time Is Delayed inSmall Intestinal Bacterial Overgrowth; J Clin Gastroenterol 49 (2015):571-576 and the review by Miazga et al.; Current views on theetiopathogenesis, clinical manifestation, diagnostics, treatment andcorrelation with other nosological entities of SIBO; Advances in MedicalSciences 60 (2015): 118-124, which highlighting reduced motility as oneof the risk factors associated with onset of SIBO.

In particular in infants and young children, proper development andgrowth of the intestinal muscles is essential for them to develop theirintestinal motility. There is thus clearly a need for alternativemethods to promote the growth of intestinal muscles, in particular toincrease the intestinal muscle thickness, such as to promote intestinalmotility and reduce the incidence of disorders associated with impairedintestinal motility, such as reduced tolerance to enteral feeding andsmall intestinal bacterial overgrowth (SIBO).

Methods to promote the intestinal muscle development and growth and topromote intestinal motility have been described in the prior art.

For example, WO 2011/65552 describes the use of a composition comprisinga hydrolysate of a protein from fermented milk (e.g. from cheese),lipids and isomaltulose to increase the muscle thickness in the smallintestine. Such compositions are however not suitable for infants andyoung children, as isomaltulose is not desirable to be used in suchcompositions.

Several studies also highlight that growth of human intestinal muscle ispromoted by insulin-like growth factor-I (see for example Kummerle etal.; IGF-I stimulates human intestinal smooth muscle cell growth byregulation of G ₁ phase cell cycle proteins; Am J Physiol GastrointestLiver Physiol 286 (2004): G412-G419). IGF-I would however be againunsuitable to be used in infant and small children diet. It would bedesirable to find suitable means to promote the growth and developmentof the intestinal muscle of infants and young children by incorporatingspecific ingredients in the infant of child's diet.

Other studies are focused on improvement of intestinal motility. Forexample, CN103372021B provides pharmaceutical composition to increasethe peristalsis in the small intestine comprising the chemicalchukrasone A. It is however desirable to avoid the use of such chemicalsfor administration to infants and young children.

Development of the intestine in infants and in young children is key forthe infant's and for the child's development in particular it is veryimportant for the infant or young child to have growth and developmentof the intestinal muscle, contributing to increased contractile capacityand intestinal motility.

It would be particularly advantageous to identify ingredients speciallyadapted to human infants and/or young children and capable of increasingintestinal muscle thickness, thus promoting the growth and/ordevelopment of the intestinal muscles and to the increase thecontractile capacity and motility, improve the enteral feeding toleranceand prevent or treat SIBO in such infants and/or young children.

Mother's milk is recommended for all infants. However, in some casesbreast feeding is inadequate or unsuccessful for medical reasons or themother chooses not to breast feed. Infant formulae have been developedfor these situations. Fortifiers have also been developed to enrichmother's milk or infant formula with specific ingredients.

There is clearly a need for developing nutritional compositions for usein methods of increasing intestinal muscle thickness, promoting thegrowth and/or development of the intestinal muscles, to the increase thecontractile capacity and motility, to promote enteral feeding toleranceand prevent and/or treat SIBO in such infants and/or young children.

There is also a need to deliver such health benefits in a manner that isparticularly suitable for the young subjects (infants and youngchildren), in a manner that does not involve a classical pharmaceuticalintervention as these infants or young children are particularlyfragile.

There is a need to deliver such health benefits in these infants oryoung children in a manner that does not induce side effects and/or in amanner that is easy to deliver, and well accepted by the parents orhealth care practitioners.

There is also a need to deliver such benefits in a manner that does keepthe cost of such delivery reasonable and affordable by most.

SUMMARY OF THE INVENTION

The present inventors have found that compositions comprising at leastone N-acetylated oligosaccharide can advantageously be used to promotethe growth and/or the development of the intestinal muscle in infantsand young children, namely by increasing the intestinal musclethickness. As a consequence, the composition comprising at least oneN-acetylated oligosaccharide can also advantageously be used to promoteintestinal contractile capacity and the intestinal motility in infantsand young children

Accordingly, the present invention therefore provides a nutritionalcomposition comprising at least one N-acetylated oligosaccharide, foruse in a method of promoting the intestinal motility in an infant or ayoung child.

The present invention also provides a growing-up milk comprising atleast one N-acetylated oligosaccharide, for use in a method of promotingthe intestinal motility in a child.

The nutritional composition, respectively the growing up milk, of thepresent invention is in particular advantageous for use in methods of

Increasing the intestinal muscle thickness,

Increasing the intestinal muscle mass,

promoting the growth and/or the development of the intestinal muscles;

Increasing the intestinal contractile capacity;

promoting the enteral feeding tolerance; or

preventing and/or treating small intestinal bacterial overgrowth (SIBO)in an infant or a young child, respectively in a child.

In a particularly advantageous embodiment, the nutritional composition,respectively the growing-up milk, comprises lacto-N-neotetraose (LNnT).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the thickness of the muscle layer of the ileum in thefollowing groups of young rats:

-   -   animals having normal in utero growth (Normal Group; positive        control)    -   animals that have experienced in utero growth retardation and        were fed a normal diet after birth (IUGR Group; negative        control),    -   animals with in-utero growth retardation having a diet after        birth supplemented with 2-FL (IUGR/2FL test group),    -   animals with in-utero growth retardation having a diet after        birth supplemented with LNnT (IUGR/LNnT test group),    -   animals with in-utero growth retardation having a diet after        birth supplemented with a combination of 2-FL and LNnT        (IUGR/HMOs mix test group).        The mean muscle thickness of each group of rats is represented        by a horizontal line. The IUGR/LNnT group shows increase of the        ileum muscle thickness over both the Normal and the IUGR groups,        whereas no increase in thickness is observed in the IUGR/2FL        group. The IUGR/HMOs Mix group showed thickness increase similar        to the IUGR/LNnT group, showing that LNnT was active even at        lower concentration and in the presence of the additional        oligosaccharide 2-FL.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms have the following meanings.

The term “infant” means a child under the age of 12 months. Theexpression “young child” means a child aged between one and less thanthree years, also called toddler. The expression “child” means a betweenthree and seven years of age.

An “infant or young child born by C-section” means an infant or youngchild who was delivered by caesarean. It means that the infant or youngchild was not vaginally delivered.

An “infant or young child vaginally born” means an infant or young childwho was vaginally delivered and not delivered by caesarean.

A “preterm” or “premature” means an infant or young child who was notborn at term. Generally it refers to an infant or young child born prior37 weeks of gestation.

An “infant having a low birth weight” means a new born having a bodyweight below 2500 g (5.5 pounds) either because of preterm birth orrestricted fetal growth. It therefore encompasses:

-   -   infant or young child who has/had a body weight from 1500 to        2500 g at birth (usually called “low birth weight” or LBW)    -   infant or young child who has/had a body weight from 1000 to        1500 g at birth (called “very low birth weight” or VLBW)    -   infant or young child who has/had a body weight under 1000 g at        birth (called “extremely low birth weight” or ELBW).

An “infant born small for gestational age (SGA)” means a baby with birthweights below the 10^(th) percentile for babies of the same gestationalage.

The expression “nutritional composition” means a composition whichnourishes a subject. This nutritional composition is usually to be takenorally or intravenously, and it usually includes a lipid or fat sourceand a protein source.

In a particular embodiment the composition of the present invention is ahypoallergenic nutritional composition. The expression “hypoallergenicnutritional composition” means a nutritional composition which isunlikely to cause allergic reactions.

In a particular embodiment the composition of the present invention is a“synthetic nutritional composition”. The expression “syntheticnutritional composition” means a mixture obtained by chemical and/orbiological means, which can be chemically identical to the mixturenaturally occurring in mammalian milks (i.e. the synthetic compositionis not breast milk).

The expression “infant formula” as used herein refers to a foodstuffintended for particular nutritional use by infants during the firstmonths of life and satisfying by itself the nutritional requirements ofthis category of person (Article 2(c) of the European CommissionDirective 91/321/EEC 2006/141/EC of 22 Dec. 2006 on infant formulae andfollow-on formulae). It also refers to a nutritional compositionintended for infants and as defined in Codex Alimentarius (Codex STAN72-1981) and Infant Specialities (incl. Food for Special MedicalPurpose). The expression “infant formula” encompasses both “starterinfant formula” and “follow-up formula” or “follow-on formula”.

A “follow-up formula” or “follow-on formula” is given from the 6th monthonwards. It constitutes the principal liquid element in theprogressively diversified diet of this category of person.

The expression “baby food” means a foodstuff intended for particularnutritional use by infants or young children during the first years oflife.

The expression “infant cereal composition” means a foodstuff intendedfor particular nutritional use by infants or young children during thefirst years of life.

The expression “growing-up milk” (or GUM) refers to a milk-based drinkgenerally with added vitamins and minerals, that is intended for youngchildren, as of one year of age or children, up to the seventh year ofage.

The term “fortifier” refers to liquid or solid nutritional compositionssuitable for mixing with breast milk or infant formula.

The expression “weaning period” means the period during which themother's milk is substituted by other food in the diet of an infant oryoung child.

The expressions “days/weeks/months/years of life” and“days/weeks/months/years of birth” can be used interchangeably.

The expression “promoting the intestinal motility” encompasses one orseveral of the following:

-   -   increasing the muscle thickness in the intestine, preferably in        the small intestine and in particular of the ileum;    -   increasing the muscle mass in the intestine, preferably in the        small intestine and in particular in the ileum;    -   promoting the growth and/or development of the intestinal        muscles, preferably of the muscles of the small intestine, in        particular the muscles of the ileum    -   promoting the intestinal contractile capacity in the intestine,        preferably in the small intestine and in particular in the        ileum;    -   promoting the enteral feeding tolerance; and    -   preventing and/or treating small intestinal bacterial overgrowth        (SIBO)

The “mother's milk” should be understood as the breast milk or thecolostrum of the mother.

An “oligosaccharide” is a saccharide polymer containing a small number(typically three to ten) of simple sugars (monosaccharides).

The term “HMO” or “HMOs” refers to human milk oligosaccharide(s). Thesecarbohydrates are highly resistant to enzymatic hydrolysis, indicatingthat they may display essential functions not directly related to theircaloric value. It has especially been illustrated that they play a vitalrole in the early development of infants and young children, such as thematuration of the immune system. Many different kinds of HMOs are foundin the human milk. Each individual oligosaccharide is based on acombination of glucose, galactose, sialic acid (N-acetylneuraminicacid), fucose and/or N-acetylglucosamine with many and varied linkagesbetween them, thus accounting for the enormous number of differentoligosaccharides in human milk—over 130 such structures have beenidentified so far. Almost all of them have a lactose moiety at theirreducing end while sialic acid and/or fucose (when present) occupyterminal positions at the non-reducing ends. The HMOs can be acidic(e.g. charged sialic acid containing oligosaccharide) or neutral (e.g.fucosylated oligosaccharide).

A “fucosylated oligosaccharide” is an oligosaccharide having a fucoseresidue. It has a neutral nature. Some examples are 2-FL(2′-fucosyllactose), 3-FL (3-fucosyllactose), difucosyllactose,lacto-N-fucopentaose (e.g. lacto-N-fucopentaose I, lacto-N-fucopentaoseII, lacto-N-fucopentaose III, lacto-N-fucopentaose V),lacto-N-fucohexaose, lacto-N-difucohexaose I, fucosyllacto-N-hexaose,fucosyllacto-N-neohexaose, difucosyllacto-N-hexaose I,difucosyllacto-N-neohexaose II and any combination thereof. Withoutwishing to be bound by theory it is believed that the fucosyl-epitope ofthe fucosylated oligosaccharides may act as decoy at the mucosalsurface. By a competition effect, it may prevent and/or limit the actionof the pathogens responsible of infections (of viral or bacterialorigin) or of their secreted components (e.g. toxins), especially byavoiding their binding to natural ligands, and without to be bound bytheory, this is believed to therefore reduce the risk ofinfections/inflammations, and particularly the risk of LRT/earinfections and/or inflammations. In addition, the fucosylatedoligosaccharides are thought to boost growth and metabolic activity ofspecific commensal microbes reducing inflammatory response and creatingan environment unfavourable for pathogens thus leading to colonizationresistance.

The expressions “fucosylated oligosaccharides comprising a2′-fucosyl-epitope” and “2-fucosylated oligosaccharides” encompassfucosylated oligosaccharides with a certain homology of form since theycontain a 2′-fucosyl-epitope, therefore a certain homology of functioncan be expected. Without wishing to be bound by theory the2′-fucosyl-epitope of these fucosylated oligosaccharides is believed tobe particularly specific to pathogens (or their secreted components)involved in the LRT and/or ear infections.

The expression “N-acetylated oligosaccharide(s)” encompasses both“N-acetyl-lactosamine” and “oligosaccharide(s) containingN-acetyl-lactosamine”. They are neutral oligosaccharides having anN-acetyl-lactosamine residue. Suitable examples are LNT(lacto-N-tetraose), para-lacto-N-neohexaose (para-LNnH), LNnT(lacto-N-neotetraose), disialyllacto-N-tetraose (DSLNT) and anycombinations thereof. Other examples are lacto-N-hexaose,lacto-N-neohexaose, para-lacto-N-hexaose, para-lacto-N-neohexaose,lacto-N-octaose, lacto-N-neooctaose, iso-lacto-N-octaose,para-lacto-N-octaose and lacto-N-decaose.

The expression “at least one fucosylated oligosaccharide” and “at leastone N-acetylated oligosaccharide” means “at least one type offucosylated oligosaccharide” and “at least one type of N-acetylatedoligosaccharide”.

A “precursor of HMO” is a key compound that intervenes in themanufacture of HMO, such as sialic acid and/or fucose.

A “sialylated oligosaccharide” is a charged sialic acid containingoligosaccharide, i.e. an oligosaccharide having a sialic acid residue.It has an acidic nature. Some examples are 3-SL (3′ sialyllactose) and6-SL (6′ sialyllactose).

The nutritional composition of the present invention can be in solidform (e.g. powder) or in liquid form. The amount of the variousingredients (e.g. the oligosaccharides) can be expressed in g/100 g ofcomposition on a dry weight basis when it is in a solid form, e.g. apowder, or as a concentration in g/L of the composition when it refersto a liquid form (this latter also encompasses liquid composition thatmay be obtained from a powder after reconstitution in a liquid such asmilk, water . . . , e.g. a reconstituted infant formula or afollow-on/follow-up formula or a growing-up milk or an infant cerealproduct or any other formulation designed for infant nutrition).

The term “prebiotic” means non-digestible carbohydrates thatbeneficially affect the host by selectively stimulating the growthand/or the activity of healthy bacteria such as bifidobacteria in thecolon of humans (Gibson G R, Roberfroid M B. Dietary modulation of thehuman colonic microbiota: introducing the concept of prebiotics. J Nutr.1995; 125:1401-12).

The term “probiotic” means microbial cell preparations or components ofmicrobial cells with a beneficial effect on the health or well-being ofthe host. (Salminen S, Ouwehand A. Benno Y. et al. “Probiotics: howshould they be defined” Trends Food Sci. Technol. 1999:10 107-10). Themicrobial cells are generally bacteria or yeasts.

The term “cfu” should be understood as colony-forming unit.

All percentages are by weight unless otherwise stated.

In addition, in the context of the invention, the terms “comprising” or“comprises” do not exclude other possible elements. The composition ofthe present invention, including the many embodiments described herein,can comprise, consist of, or consist essentially of the essentialelements and limitations of the invention described herein, as well asany additional or optional ingredients, components, or limitationsdescribed herein or otherwise depending on the needs.

Any reference to prior art documents in this specification is not to beconsidered an admission that such prior art is widely known or formspart of the common general knowledge in the field.

The invention will now be described in further details. It is noted thatthe various aspects, features, examples and embodiments described in thepresent application may be compatible and/or combined together.

A first object of the present invention is therefore a nutritionalcomposition comprising at least one N-acetylated oligosaccharide, foruse in a method of promoting the intestinal motility in an infant or ayoung child. Preferably, the method is a method of promoting themotility in the small intestine, preferably in the ileum, of an infantor a young child.

A second object of the present invention is a growing-up milk comprisingat least one N-acetylated oligosaccharide, for use in a method ofpromoting the intestinal motility in a child. Preferably, the method isa method of promoting the motility in the small intestine, preferably inthe ileum, of a child.

Preferably, the nutritional composition, respectively the growing-upmilk, of the present invention is for use in a method of promoting theintestinal contractile capacity in an infant or a young child,respectively in a child. More preferably, the method is a method ofpromoting the contractile capacity in the small intestine, morepreferably in the ileum.

Preferably, the nutritional composition, respectively the growing-upmilk, of the present invention is for use in a method of promoting thegrowth and/or the development of the intestinal muscles in an infant ora young child, respectively in a child. More preferably, the method is amethod of promoting the growth and/or the development of the intestinalmuscles in the small intestine, even more preferably in the ileum. In apreferred aspect the muscle is smooth muscle.

To achieve such benefits, the nutritional composition, respectively thegrowing-up milk, of the invention is preferably for use in a method ofincreasing the intestinal muscle mass, preferably the intestinal musclethickness in an infant or a young child, respectively in a child. Morepreferably, it is a method of increasing the mass, preferably thethickness of the muscle in the small intestine, even more preferably inthe ileum. In a preferred aspect the muscle is smooth muscle.

As a consequence of the improvement of motility, the nutritionalcomposition, respectively the growing-up milk, of the invention ispreferably for use in a method of promoting enteral feeding tolerance inan infant or a young child, respectively in a child.

Also as a consequence of the improvement of motility, the nutritionalcomposition, respectively the growing-up milk, of the invention ispreferably for use in a method of preventing and/or treating Smallintestinal bacterial overgrowth (SIBO) in an infant or a young child,respectively in a child.

The nutritional composition, respectively the growing-up milk, of thepresent invention comprises at least one the N-acetylatedoligosaccharide. There can be one or several types of N-acetylatedoligosaccharide. The N-acetylated oligosaccharide(s) can be for examplelacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT) or any combinationthereof. In some particular embodiment the N-acetylated oligosaccharideis lacto-N-neotetraose (LNnT), para-lacto-N-neohexaose (para-LNnH),disialyllacto-N-tetraose (DSLNT) or any combination thereof. In someparticular embodiments the N-acetylated oligosaccharide is LNnT. In someparticular embodiments the N-acetylated oligosaccharide is LNT. In someother particular embodiments the N-acetylated oligosaccharide is amixture of LNT and LNnT. In some particular embodiments the nutritionalcomposition or the growing-up milk comprises both LNT and LNnT in aratio LNT:LNnT between 5:1 and 1:2, or from 2:1 to 1:1, or from 2:1.2 to2:1.6.

In a preferred embodiment, the nutritional composition or the growing-upmilk according to the invention comprises lacto-N-neotetraose (LNnT). Ina particular embodiment, there is no other type of N-acetylatedoligosaccharide than lacto-N-neotetraose (LNnT), i.e. the nutritionalcomposition or the growing-up milk of the invention comprises onlylacto-N-neotetraose (LNnT) as N-acetylated oligosaccharide.

The N-acetylated oligosaccharide(s) may be synthesised chemically byenzymatic transfer of saccharide units from donor moieties to acceptormoieties using glycosyltransferases as described for example in U.S.Pat. No. 5,288,637 and WO 96/10086. Alternatively, LNT and LNnT may beprepared by chemical conversion of Keto-hexoses (e.g. fructose) eitherfree or bound to an oligosaccharide (e.g. lactulose) intoN-acetylhexosamine or an N-acetylhexosamine-containing oligosaccharideas described in Wrodnigg, T. M.; Stutz, A. E. (1999) Angew. Chem. Int.Ed. 38:827-828. N-acetyl-lactosamine produced in this way may then betransferred to lactose as the acceptor moiety.

In a particularly advantageous embodiment of the present invention, theN-acetylated oligosaccharide(s) are present in the nutritionalcomposition or the growing-up milk in some particular amounts. The term“amount” refers to the total amount of each of these two components inthe nutritional composition or the growing-up milk unless otherwisespecified. It therefore does not refer to an individual amount exceptwhen there is a single type of these components (in that case both thetotal and individual amounts equal). By way of illustrative example, ifthere is only one (i.e. only one type of) N-acetylated oligosaccharidein the composition (e.g. LNnT), its individual amount (and therefore thetotal amount of N-acetylated oligosaccharides) will be in the range0.75-1.65 g/L. If there are several (i.e. several types of) N-acetylatedoligosaccharides, their individual amount will be lower (e.g. if thereare 2 different types of N-acetylated oligosaccharide, e.g. LNnT+LNT,there may be for example each in an individual amount of 0.5 g/L) butthe total amount of N-acetylated oligosaccharides will be in the range0.75-1.65 g/L.

The N-acetylated oligosaccharide(s) can be present in the nutritionalcomposition or the growing-up milk according to the present invention ina total amount of 0.1-3 g/L of the composition. In some embodiments, theN-acetylated oligosaccharide(s) may be in a total amount of 0.01-2.5 g/Lof the composition, such as 0.05-1.5 g/L or 0.05-1 g/L or 0.05-0.8 g/Lor 0.08-0.8 g/L or 0.09-0.8 g/L of the composition. In a particularembodiment, the N-acetylated oligosaccharide(s) is/are in a total amountof 0.5 g/L of the composition. In another particular embodiment, theN-acetylated oligosaccharide(s) is/are in a total amount of 0.13 g/L ofthe composition.

The N-acetylated oligosaccharide(s) can be present in the nutritionalcomposition or the growing-up milk in a total amount of 0.008-2.3 g/100g of composition on a dry weight basis. The N-acetylatedoligosaccharide(s) may be in a total amount of 0.008-1.9 g/100 g ofcomposition, such as 0.04-1.2 g/100 g or 0.04-0.8 g/100 g or 0.04-0.6g/100 g or 0.06-0.6 g/100 g or 0.07-0.6 g/100 g. In a particularembodiment, the N-acetylated oligosaccharide(s) is/are in a total amountof 0.38 g/100 g of the composition. In another particular embodiment,the N-acetylated oligosaccharide(s) is/are in a total amount of 0.1g/100 g of the composition.

In a particular embodiment, the N-acetylated oligosaccharide is providedin the nutritional composition or growing-up milk of the presentinvention in such an amount that normal consumption of the nutritionalcomposition or growing-up milk would provide to the infant or youngchild, respectively the child, consuming it a total daily dose of0.003-3.9 g, preferably 0.006-3.25 g or 0.03-1.95 g or 0.03-1.3 g or0.03-1 g, for example 0.05-1 g per day.

For preterm, low birth weight and small for gestational age infants, thedaily dose is preferably of 0.005 to 0.1 g/kg of body weight per day,preferably 0.006-0.09 g or 0.007-0.08 g or 0.008-0.07 g or 0.009-0.06 gor 0.01-0.05 g or 0.02-0.04 g/, most preferably 0.034 g per kg of bodyweight and per day.

The nutritional composition or the growing-up milk according to thepresent invention may also comprise at least another oligosaccharide(s)(i.e. other than the N-acetylated oligosaccharide(s) necessarily presentin the composition) and/or at least a fiber(s) and/or at least aprecursor(s) thereof. The other oligosaccharide and/or fiber and/orprecursor thereof may be selected from the list comprising fucosylatedoligosaccharides, galacto-oligosaccharides (GOS),fructo-oligosaccharides (FOS), inulin, xylooligosaccharides (XOS),polydextrose, sialylated oligosaccharides, sialic acid, fucose and anycombination thereof. They may be in an amount between 0 and 10% byweight of composition. In a particular embodiment, the nutritionalcomposition or the growing-up milk can also contain at least one BMO(bovine milk oligosaccharide).

Suitable commercial products that can be used in addition to theoligosaccharides comprised in the oligosaccharide mixture to prepare thenutritional compositions or the growing-up milk according to theinvention include combinations of FOS with inulin such as the productsold by BENEO under the trademark Orafti, or polydextrose sold by Tate &Lyle under the trademark STA-LITE®.

In a particular embodiment, the nutritional composition or thegrowing-up milk of the present invention may contain additional humanmilk oligosaccharides. For example, it may comprise at least onefucosylated oligosaccharide. There can be one or several types offucosylated oligosaccharide(s). The fucosylated oligosaccharide(s) canindeed be selected from the list comprising 2′-fucosyllactose,3′fucosyllactose, difucosyllactose, lacto-N-fucopentaose (such aslacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaoseIII, lacto-N-fucopentaose V), lacto-N-fucohexaose, lacto-N-difucohexaoseI, fucosyllacto-N-hexaose, fucosyllacto-N-neohexaose (such asfucosyllacto-N-neohexaose I, fucosyllacto-N-neohexaose II),difucosyllacto-N-hexaose I, difuco-lacto-N-neohexaose,difucosyllacto-N-neohexaose I, difucosyllacto-N-neohexaose II,fucosyl-para-Lacto-N-hexaose, tri-fuco-para-Lacto-N-hexaose I and anycombination thereof.

In some particular embodiments the fucosylated oligosaccharide comprisesa 2′-fucosyl-epitope. It can be for example selected from the listcomprising 2′-fucosyllactose, difucosyllactose, lacto-N-fucopentaose,lacto-N-fucohexaose, lacto-N-difucohexaose, fucosyllacto-N-hexaose,fucosyllacto-N-neohexaose, difucosyllacto-N-hexaosedifuco-lacto-N-neohexaose, difucosyllacto-N-neohexaose,fucosyl-para-Lacto-N-hexaose and any combination thereof.

In a particular embodiment, the nutritional composition, respectivelythe growing-up milk, according to the invention comprises2′-fucosyllactose (or 2FL, or 2′FL, or 2-FL or 2′-FL). In a particularembodiment, there is no other type of fucosylated oligosaccharide than2′-fucosyllactose, i.e. the nutritional composition or growing-up milkof the invention comprises only 2′-fucosyllactose as fucosylatedoligosaccharide.

The fucosylated oligosaccharide(s) may be isolated by chromatography orfiltration technology from a natural source such as animal milks.Alternatively, it may be produced by biotechnological means usingspecific fucosyltransferases and/or fucosidases either through the useof enzyme-based fermentation technology (recombinant or natural enzymes)or microbial fermentation technology. In the latter case, microbes mayeither express their natural enzymes and substrates or may be engineeredto produce respective substrates and enzymes. Single microbial culturesand/or mixed cultures may be used. Fucosylated oligosaccharide formationcan be initiated by acceptor substrates starting from any degree ofpolymerization (DP), from DP=1 onwards. Alternatively, fucosylatedoligosaccharides may be produced by chemical synthesis from lactose andfree fucose. Fucosylated oligosaccharides are also available for examplefrom Kyowa, Hakko, Kogyo of Japan.

When present, the fucosylated oligosaccharide(s) can be in thenutritional composition or the growing-up milk according to the presentinvention in a total amount of 0.75-1.65 g/L of the composition. In someembodiments, the fucosylated oligosaccharide(s) may be in a total amountof 0.005-5 g/L of the composition, such as 0.01-3 g/L or 0.02-2 g/L or0.1-2.5 g/L or 0.15-2 g/L or 0.25-1.9 g/L of the composition. In aparticular embodiment, the fucosylated oligosaccharide(s) is/are in atotal amount of 1 g/L of the composition. In another particularembodiment, the fucosylated oligosaccharide(s) is/are in a total amountof 0.26 g/L of the composition.

The fucosylated oligosaccharide(s) can be present in the nutritionalcomposition or the growing-up milk in a total amount of 0.004-3.8 g/100g of composition on a dry weight basis. The fucosylatedoligosaccharide(s) may be in a total amount of 0.008-2.3 g/100 g of thecomposition, such as 0.015-1.5 g/100 g, or 0.08-1.9 g/100 g or 0.12-1.5g/100 g or 0.15-1.5 g/100 g or 0.19-1.5 g/100 g of the composition. In aparticular embodiment, the fucosylated oligosaccharide(s) is/are in atotal amount of 0.78 g/100 g of the composition. In another particularembodiment, the fucosylated oligosaccharide(s) is/are in a total amountof 0.2 g/100 g of the composition.

In a particular embodiment, the fucosylated oligosaccharide is providedin the nutritional composition or growing-up milk of the presentinvention in such an amount that normal consumption of the nutritionalcomposition or growing-up milk would provide to the infant or youngchild, respectively the child, consuming it a total daily dose of0.003-6.5 g, preferably 0.006-3.9 g, for example 0.012-2.6 g per day.

For preterm, low birth weight and small for gestational age infants, thedaily dose of fucosylated oligosaccharides is preferably of 0.05 to 1g/kg of body weight per day, preferably 0.06-0.9 g or 0.07-0.8 g or0.08-0.7 g or 0.09-0.6 g or 0.1-0.5 g or 0.2-0.4 g/, most preferably0.34 g per kg of body weight and per day.

When fucosylated oligosaccharides are present, the fucosylatedoligosaccharide(s) and the N-acetylated oligosaccharide(s) comprised inthe nutritional composition or the growing-up milk according to theinvention are typically present in a N-acetylatedoligosaccharide(s):fucosylated oligosaccharide(s) of from 1:20 to 2:1,preferably 1:15 to 1:1, most preferably of 1:10 to 1:2. In aparticularly advantageous embodiment, this ratio is 2:1 or around 2:1.

In a particular embodiment of the present invention, the nutritionalcomposition or the growing-up milk comprises 2′-fucosyllactose (2FL) andlacto-N-neotetraose (LNnT).

In another specific embodiment, the nutritional composition or thegrowing-up milk of the present invention comprises an oligosaccharidemixture that consists of 2′-fucosyllactose (2-FL) andlacto-N-neotetraose (LNnT). In other words, the nutritional compositionor the growing-up milk of the invention comprises only 2′-fucosyllactose(2-FL) as fucosylated oligosaccharide and only lacto-N-neotetraose(LNnT) as N-acetylated oligosaccharide.

In another particular embodiment, the nutritional composition or thegrowing-up milk according to the invention can comprise sialylatedoligosaccharide(s). There can be one or several sialylatedoligosaccharide(s). The sialylated oligosaccharide(s) can be selectedfrom the group comprising 3′ sialyllactose (3-SL), 6′ sialyllactose(6-SL), and any combination thereof. In some embodiments of theinvention the composition comprises 3-SL and 6-SL. In some particularembodiments the ratio between 3′-sialyllactose (3-SL) and6′-sialyllactose (6-SL) can be in the range between 5:1 and 1:10, orfrom 3:1 and 1:1, or from 1:1 to 1:10. In some specific embodiments thesialylated oligosaccharide of the composition is 6′ sialyllactose(6-SL).

The sialylated oligosaccharide(s) may be isolated by chromatographic orfiltration technology from a natural source such as animal milks.Alternatively, they may be produced by biotechnological means usingspecific sialyltransferases or sialidases, neuraminidases, either by anenzyme based fermentation technology (recombinant or natural enzymes),by chemical synthesis or by a microbial fermentation technology. In thelatter case microbes may either express their natural enzymes andsubstrates or may be engineered to produce respective substrates andenzymes. Single microbial cultures or mixed cultures may be used.Sialyl-oligosaccharide formation can be initiated by acceptor substratesstarting from any degree of polymerisation (DP), from DP=1 onwards.Alternatively, sialyllactoses may be produced by chemical synthesis fromlactose and free N′-acetylneuraminic acid (sialic acid). Sialyllactosesare also commercially available for example from Kyowa Hakko Kogyo ofJapan.

In another preferred embodiment of the invention the nutritionalcomposition or the growing-up milk may comprise from 0.005-5 g/L ofsialylated oligosaccharides, or 0.008-2.5 g/L, or 0.01-1 g/L, or0.02-0.7 g/L, for example 0.03-0.5 g/L.

The nutritional composition or the growing-up milk according to theinvention can contain 0.004-3.8 g of sialylated oligosaccharides per 100g of composition on a dry weight basis, e.g. 0.006-1.9 g or 0.008-0.8 gor 0.015-0.5 g, for example 0.023-0.4 of sialylated oligosaccharides per100 g of composition on a dry weight basis.

In some particular embodiments of the present invention, the nutritionalcomposition or the growing-up milk comprises sialylatedoligosaccharide(s) in an amount of below 0.1g/100 g of composition on adry weight basis.

In a particular embodiment, the sialylated oligosaccharide is providedin the nutritional composition or growing-up milk of the presentinvention in such an amount that normal consumption of the nutritionalcomposition or growing-up milk would provide to the infant or youngchild, respectively the child, consuming it a total daily dose of0.003-6.5 g, preferably 0.005-3.3 g or 0.006-1.3 g or 0.01-0.9 g, forexample 0.018-0.65 g per day.

In some particular embodiments of the present invention, the nutritionalcomposition or the growing-up milk does not contain any sialylatedoligosaccharide(s).

The nutritional composition or the growing-up milk according to thepresent invention may optionally also comprise at least one precursor ofoligosaccharide. There can be one or several precursor(s) ofoligosaccharide. For example the precursor of human milk oligosaccharideis sialic acid, fucose or a mixture thereof. In some particularembodiments the composition comprises sialic acid.

In particular examples the nutritional composition or the growing-upmilk comprises from 0 to 3 g/L of precursor(s) of oligosaccharide, orfrom 0 to 2 g/L, or from 0 to 1 g/L, or from 0 to 0.7 g/L, or from 0 to0.5 g/L or from 0 to 0.3 g/L, or from 0 to 0.2 g/L of precursor(s) ofoligosaccharide. The composition according to the invention can containfrom 0 to 2.1 g of precursor(s) of oligosaccharide per 100 g ofcomposition on a dry weight basis, e.g. from 0 to 1.5 g or from 0 to 0.8g or from 0 to 0.15 g of precursor(s) of oligosaccharide per 100 g ofcomposition on a dry weight basis.

The nutritional composition or the growing-up milk of the presentinvention can further comprise at least one probiotic (or probioticstrain), such as a probiotic bacterial strain.

The probiotic microorganisms most commonly used are principally bacteriaand yeasts of the following genera: Lactobacillus spp., Streptococcusspp., Enterococcus spp., Bifidobacterium spp. and Saccharomyces spp.

In some particular embodiments, the probiotic is a probiotic bacterialstrain. In some specific embodiments, it is particularly Bifidobacteriaand/or Lactobacilli.

Suitable probiotic bacterial strains include Lactobacillus rhamnosusATCC 53103 available from Valio Oy of Finland under the trademark LGG,Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus paracasei CNCM1-2116, Lactobacillus johnsonii CNCM 1-1225, Streptococcus salivariusDSM 13084 sold by BLIS Technologies Limited of New Zealand under thedesignation K12, Bifidobacterium lactis CNCM 1-3446 sold inter alia bythe Christian Hansen company of Denmark under the trademark Bb 12,Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co.Ltd. of Japan under the trademark BB536, Bifidobacterium breve sold byDanisco under the trademark Bb-03, Bifidobacterium breve sold byMorinaga under the trade mark M-16V, Bifidobacterium infantis sold byProcter & Gamble Co. under the trademark Bifantis and Bifidobacteriumbreve sold by Institut Rosell (Lallemand) under the trademark R0070.

The nutritional composition or the growing-up milk according to theinvention may contain from 10e3 to 10e12 cfu of probiotic strain, morepreferably between 10e7 and 10e12 cfu such as between 10e8 and 10e10 cfuof probiotic strain per g of composition on a dry weight basis.

In one embodiment the probiotics are viable. In another embodiment theprobiotics are non-replicating or inactivated. There may be both viableprobiotics and inactivated probiotics in some other embodiments.Probiotic components and metabolites can also be added.

The nutritional composition according to the invention can be forexample an infant formula, a starter infant formula, a follow-on orfollow-up formula, a growing-up milk, a baby food, an infant cerealcomposition, a fortifier such as a human milk fortifier, or asupplement. In some particular embodiments, the composition of theinvention is an infant formula, a fortifier or a supplement that may beintended for the first 4 or 6 months of age. In a preferred embodimentthe nutritional composition of the invention is an infant formula.

In some other embodiments the nutritional composition of the presentinvention is a fortifier. The fortifier can be a breast milk fortifier(e.g. a human milk fortifier) or a formula fortifier such as an infantformula fortifier or a follow-on/follow-up formula fortifier.

When the nutritional composition is a supplement, it can be provided inthe form of unit doses. In such cases it is particularly useful todefine the amount of N-acetylated oligosaccharides and optionally otheroligosaccharides in terms or daily dose to be administered to the infantor young child, such as described above.

The nutritional composition of the present invention can be in solid(e.g. powder), liquid or gelatinous form.

In a specific embodiment the nutritional composition is a supplement inpowder form and provided in a sachet, or in the form of a syrup. Whenthe supplement is in powder form, it may comprise a carrier. It ishowever preferred that the supplement is devoid of a carrier. When thesupplement is in the form of a syrup, the HMOs are preferably dissolvedor suspended in water acidified with citrate.

The nutritional composition or the growing-up milk according to theinvention generally contains a protein source. The protein can be in anamount of from 1.6 to 3 g per 100 kcal. In some embodiments, especiallywhen the composition is intended for premature infants, the proteinamount can be between 2.4 and 4 g/100 kcal or more than 3.6 g/100 kcal.In some other embodiments the protein amount can be below 2.0 g per 100kcal, e.g. between 1.8 to 2 g/100 kcal, or in an amount below 1.8 g per100 kcal.

The type of protein is not believed to be critical to the presentinvention provided that the minimum requirements for essential aminoacid content are met and satisfactory growth is ensured. Thus, proteinsources based on whey, casein and mixtures thereof may be used as wellas protein sources based on soy. As far as whey proteins are concerned,the protein source may be based on acid whey or sweet whey or mixturesthereof and may include alpha-lactalbumin and beta-lactoglobulin in anydesired proportions.

In some advantageous embodiments the protein source is whey predominant(i.e. more than 50% of proteins are coming from whey proteins, such as60% or 70%).

The proteins may be intact or hydrolysed or a mixture of intact andhydrolysed proteins. By the term “intact” is meant that the main part ofthe proteins are intact, i.e. the molecular structure is not altered,for example at least 80% of the proteins are not altered, such as atleast 85% of the proteins are not altered, preferably at least 90% ofthe proteins are not altered, even more preferably at least 95% of theproteins are not altered, such as at least 98% of the proteins are notaltered. In a particular embodiment, 100% of the proteins are notaltered.

The term “hydrolysed” means in the context of the present invention aprotein which has been hydrolysed or broken down into its componentamino acids. The proteins may be either fully or partially hydrolysed.It may be desirable to supply partially hydrolysed proteins (degree ofhydrolysis between 2 and 20%), for example for infants or young childrenbelieved to be at risk of developing cow's milk allergy. If hydrolysedproteins are required, the hydrolysis process may be carried out asdesired and as is known in the art. For example, whey proteinhydrolysates may be prepared by enzymatically hydrolysing the wheyfraction in one or more steps. If the whey fraction used as the startingmaterial is substantially lactose free, it is found that the proteinsuffers much less lysine blockage during the hydrolysis process. Thisenables the extent of lysine blockage to be reduced from about 15% byweight of total lysine to less than about 10% by weight of lysine; forexample about 7% by weight of lysine which greatly improves thenutritional quality of the protein source.

In an embodiment of the invention at least 70% of the proteins arehydrolysed, preferably at least 80% of the proteins are hydrolysed, suchas at least 85% of the proteins are hydrolysed, even more preferably atleast 90% of the proteins are hydrolysed, such as at least 95% of theproteins are hydrolysed, particularly at least 98% of the proteins arehydrolysed. In a particular embodiment, 100% of the proteins arehydrolysed.

In one particular embodiment the proteins of the nutritional compositionare hydrolyzed, fully hydrolyzed or partially hydrolyzed. The degree ofhydrolysis (DH) of the protein can be between 8 and 40, or between 20and 60 or between 20 and 80 or more than 10, 20, 40, 60, 80 or 90.

The protein component can alternatively be replaced by a mixture of freeamino acid, for example for preterm or low birth weight infants. Thefree amino acids can be obtained by complete hydrolysis of proteins (DHof 100) or can be synthetic amino acids.

In a particular embodiment the nutritional composition or the growing-upmilk according to the invention is a hypoallergenic composition. Inanother particular embodiment the composition according to the inventionis a hypoallergenic nutritional composition or growing-up milk.

The nutritional composition or the growing-up milk according to thepresent invention generally contains a carbohydrate source. This isparticularly preferable in the case where the nutritional composition ofthe invention is an infant formula. In this case, any carbohydratesource conventionally found in infant formulae such as lactose, sucrose,saccharose, maltodextrin, starch and mixtures thereof may be usedalthough one of the preferred sources of carbohydrates is lactose.

The nutritional composition or the growing-up milk according to thepresent invention generally contains a source of lipids. This isparticularly relevant if the nutritional composition of the invention isan infant formula. In this case, the lipid source may be any lipid orfat which is suitable for use in infant formulae. Some suitable fatsources include palm oil, structured triglyceride oil, high oleicsunflower oil and high oleic safflower oil, medium-chain-triglycerideoil. The essential fatty acids linoleic and α-linolenic acid may also beadded, as well small amounts of oils containing high quantities ofpreformed arachidonic acid and docosahexaenoic acid such as fish oils ormicrobial oils. The fat source may have a ratio of n-6 to n-3 fattyacids of about 5:1 to about 15:1; for example about 8:1 to about 10:1.

The nutritional composition or the growing-up milk of the invention mayalso contain all vitamins and minerals understood to be essential in thedaily diet and in nutritionally significant amounts. Minimumrequirements have been established for certain vitamins and minerals.Examples of minerals, vitamins and other nutrients optionally present inthe composition of the invention include vitamin A, vitamin B1, vitaminB2, vitamin B6, vitamin B12, vitamin E, vitamin K, vitamin C, vitamin D,folic acid, inositol, niacin, biotin, pantothenic acid, choline,calcium, phosphorous, iodine, iron, magnesium, copper, zinc, manganese,chlorine, potassium, sodium, selenium, chromium, molybdenum, taurine,and L-carnitine. Minerals are usually added in salt form. The presenceand amounts of specific minerals and other vitamins will vary dependingon the intended population.

If necessary, the nutritional composition or the growing-up milk of theinvention may contain emulsifiers and stabilisers such as soy, lecithin,citric acid esters of mono- and diglycerides, and the like.

The nutritional composition or the growing-up milk of the invention mayalso contain other substances which may have a beneficial effect such aslactoferrin, nucleotides, nucleosides, and the like.

The nutritional composition or the growing-up milk of the invention mayalso contain carotenoid(s). In some particular embodiments of theinvention, the nutritional composition of the invention does notcomprise any carotenoid.

The nutritional composition or the growing-up milk according to theinvention may be prepared in any suitable manner. A composition will nowbe described by way of example.

For example, a formula such as an infant formula may be prepared byblending together the protein source, the carbohydrate source and thefat source in appropriate proportions. If used, the emulsifiers may beincluded at this point. The vitamins and minerals may be added at thispoint but they are usually added later to avoid thermal degradation. Anylipophilic vitamins, emulsifiers and the like may be dissolved into thefat source prior to blending. Water, preferably water which has beensubjected to reverse osmosis, may then be mixed in to form a liquidmixture. The temperature of the water is conveniently in the rangebetween about 50° C. and about 80° C. to aid dispersal of theingredients. Commercially available liquefiers may be used to form theliquid mixture.

The fucosylated oligosaccharide(s) and the N-acetylatedoligosaccharide(s) may be added at this stage, especially if the finalproduct is to have a liquid form. If the final product is to be apowder, they may likewise be added at this stage if desired.

The liquid mixture is then homogenised, for example in two stages.

The liquid mixture may then be thermally treated to reduce bacterialloads, by rapidly heating the liquid mixture to a temperature in therange between about 80° C. and about 150° C. for a duration betweenabout 5 seconds and about 5 minutes, for example. This may be carriedout by means of steam injection, an autoclave or a heat exchanger, forexample a plate heat exchanger.

Then, the liquid mixture may be cooled to between about 60° C. and about85° C. for example by flash cooling. The liquid mixture may then beagain homogenised, for example in two stages between about 10 MPa andabout 30 MPa in the first stage and between about 2 MPa and about 10 MPain the second stage. The homogenised mixture may then be further cooledto add any heat sensitive components, such as vitamins and minerals. ThepH and solids content of the homogenised mixture are convenientlyadjusted at this point.

If the final product is to be a powder, the homogenised mixture istransferred to a suitable drying apparatus such as a spray dryer orfreeze dryer and converted to powder. The powder should have a moisturecontent of less than about 5% by weight. The fucosylatedoligosaccharide(s) and the N-acetylated oligosaccharide(s) may also oralternatively be added at this stage by dry-mixing or by blending themin a syrup form of crystals, along with the probiotic strain(s) (ifused), and the mixture is spray-dried or freeze-dried.

If a liquid composition is preferred, the homogenised mixture may besterilised then aseptically filled into suitable containers or may befirst filled into the containers and then retorted.

In another embodiment, the composition of the invention may be asupplement. The supplement may be in the form of tablets, capsules,pastilles or a liquid for example. The supplement may further containprotective hydrocolloids (such as gums, proteins, modified starches),binders, film forming agents, encapsulating agents/materials, wall/shellmaterials, matrix compounds, coatings, emulsifiers, surface activeagents, solubilizing agents (oils, fats, waxes, lecithins etc.),adsorbents, carriers, fillers, co-compounds, dispersing agents, wettingagents, processing aids (solvents), flowing agents, taste maskingagents, weighting agents, jellifying agents and gel forming agents. Thesupplement may also contain conventional pharmaceutical additives andadjuvants, excipients and diluents, including, but not limited to,water, gelatine of any origin, vegetable gums, lignin-sulfonate, talc,sugars, starch, gum arabic, vegetable oils, polyalkylene glycols,flavouring agents, preservatives, stabilizers, emulsifying agents,buffers, lubricants, colorants, wetting agents, fillers, and the like.

Further, the supplement may contain an organic or inorganic carriermaterial suitable for oral or parenteral administration as well asvitamins, minerals trace elements and other micronutrients in accordancewith the recommendations of Government bodies such as the USRDA.

The nutritional composition according to the invention is for use ininfants or young children. The infants or young children may be bornterm or preterm. In a particular embodiment the nutritional compositionof the invention is for use in infants or young children that were bornpreterm, having a low birth weight and/or born small for gestational age(SGA). In a particular embodiment the nutritional composition of theinvention is for use in preterm infants, infants having a low birthweight and/or infants born small for gestational age (SGA).

The nutritional composition of the present invention may also be used inan infant or a young child that was born by C-section or that wasvaginally delivered.

In some embodiments the composition according to the invention can befor use before and/or during the weaning period.

The nutritional composition can be administered (or given or fed) at anage and for a period that depends on the needs.

The nutritional composition can be for example given immediately afterbirth of the infants. The composition of the invention can also be givenduring the first week of life of the infant, or during the first 2 weeksof life, or during the first 3 weeks of life, or during the first monthof life, or during the first 2 months of life, or during the first 3months of life, or during the first 4 months of life, or during thefirst 6 months of life, or during the first 8 months of life, or duringthe first 10 months of life, or during the first year of life, or duringthe first two years of life or even more. In some particularlyadvantageous embodiments of the invention, the nutritional compositionis given (or administered) to an infant within the first 4, 6 or 12months of birth of said infant. In some other embodiments, thenutritional composition of the invention is given few days (e.g. 1, 2,3, 5, 10, 15, 20 . . . ), or few weeks (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10 . . . ), or few months (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . )after birth. This may be especially the case when the infant ispremature, but not necessarily.

In one embodiment the composition of the invention is given to theinfant or young child as a supplementary composition to the mother'smilk. In some embodiments the infant or young child receives themother's milk during at least the first 2 weeks, first 1, 2, 4, or 6months. In one embodiment the nutritional composition of the inventionis given to the infant or young child after such period of mother'snutrition, or is given together with such period of mother's milknutrition. In another embodiment the composition is given to the infantor young child as the sole or primary nutritional composition during atleast one period of time, e.g. after the 1^(st), 2^(nd) or 4^(th) monthof life, during at least 1, 2, 4 or 6 months.

In one embodiment the nutritional composition of the invention is acomplete nutritional composition (fulfilling all or most of thenutritional needs of the subject). In another embodiment the nutritioncomposition is a supplement or a fortifier intended for example tosupplement human milk or to supplement an infant formula or afollow-on/follow-up formula.

EXAMPLES

The following examples illustrate some specific embodiments of thecomposition for use according to the present invention. The examples aregiven solely for the purpose of illustration and are not to be construedas limitations of the present invention.

Example 1

An example of the composition of a nutritional composition (e.g. aninfant formula) according to the present invention is given in the belowtable 1. This composition is given by way of illustration only.

TABLE 1 Composition of the infant formula of Example 1 Nutrients per 100kcal per litre Energy (kcal) 100 670 Protein (g) 1.83 12.3 Fat (g) 5.335.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg) 101 675 Lactose(g) 11.2 74.7 Minerals (g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg)64 430 Ca (mg) 62 410 P (mg) 31 210 Mg (mg) 7 50 Mn (μg) 8 50 Se (μg) 213 Vitamin A (μg RE) 105 700 Vitamin D (μg) 1.5 10 Vitamin E (mg TE) 0.85.4 Vitamin K1 (μg) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47Vitamin B2 (mg) 0.15 1.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50Folic acid (μg) 9 60 Pantothenic acid (mg) 0.45 3 Vitamin B12 (μg) 0.3 2Biotin (μg) 2.2 15 Choline (mg) 10 67 Fe (mg) 1.2 8 I (μg) 15 100 Cu(mg) 0.06 0.4 Zn (mg) 0.75 5 Oligosaccharides LNnT (g) 0.075 0.5 (HMOs)

Example 2

An example of the composition of a nutritional composition (e.g. aninfant formula) according to the present invention is given in the belowtable 2. This composition is given by way of illustration only.

TABLE 2 Composition of the infant formula of Example 2 Nutrients per 100kcal per litre Energy (kcal) 100 670 Protein (g) 1.83 12.3 Fat (g) 5.335.7 Linoleic acid (g) 0.79 5.3 α-Linolenic acid (mg) 101 675 Lactose(g) 11.2 74.7 Minerals (g) 0.37 2.5 Na (mg) 23 150 K (mg) 89 590 Cl (mg)64 430 Ca (mg) 62 410 P (mg) 31 210 Mg (mg) 7 50 Mn (μg) 8 50 Se (μg) 213 Vitamin A (μg RE) 105 700 Vitamin D (μg) 1.5 10 Vitamin E (mg TE) 0.85.4 Vitamin K1 (μg) 8 54 Vitamin C (mg) 10 67 Vitamin B1 (mg) 0.07 0.47Vitamin B2 (mg) 0.15 1.0 Niacin (mg) 1 6.7 Vitamin B6 (mg) 0.075 0.50Folic acid (μg) 9 60 Pantothenic acid (mg) 0.45 3 Vitamin B12 (μg) 0.3 2Biotin (μg) 2.2 15 Choline (mg) 10 67 Fe (mg) 1.2 8 I (μg) 15 100 Cu(mg) 0.06 0.4 Zn (mg) 0.75 5 Oligosaccharides 2FL (g) 0.15 1 (HMOs) LNnT(g) 0.075 0.5

Example 3 Description of the Study

Three groups of time-mated pregnant Sprague-Dawley female rats werebought from Charles River laboratories. One group was submitted to foodrestriction of 60% during the last 10 days of gestation and theiroffspring were cross-fostered to normally fed rats. A second group ofpregnant females was normally fed and their offspring cross-fosteredamong the same group of dams. Immediately after birth (postnatal day 2(d=2)), the born rat pups—subjects of the experiment—were assigned toone of the following groups:

-   Normal group (positive control): Normal-weight offspring born from    unrestricted dams and fed after birth a normal non-supplemented    diet;-   IUGR group (negative control; n=20): IUGR rats being fed a normal    diet after birth;-   IUGR rats+HMO (test groups): During the experiment, they were reared    by their mothers for 21 days and supplemented with HMOs (2-FL, LNnT    or the Mix of 2-FL and LNnT). At weaning, they were fed a diet    supplemented with the same HMOs, see details below. There were three    different groups:    -   IUGR/2FL group (n=10): supplemented with 2-FL only;    -   IUGR/LNnT group (n=10): supplemented with LNnT only; and    -   IUGR/HMOs mix group (n=10): supplemented with LNnT and 2FL in a        weight ratio of 1:2.

All rat pups were fed from birth (d =1) up to 57 days (d =57) based onthe following protocol:

-   d1 to d6: all groups of rats were nursed by dam (mothers' milk    only);-   d7 to d21: all rats were nursed by dam (mothers' milk only). But in    the test groups, 3 g/kg body weight of HMO were also administered by    gavage:    -   3 g/kg body weight of 2-FL, for the IUGR/2FL goup;    -   3 g/kg body weight of LNnT, for the IUGR/LNnT group;    -   3 g/kg body weight of a mix of LNnT and 2FL at a 1:2 weight        ratio, for the IUGR/HMOs mix group;-   d22 to d57: all rats were fed a diet as detailed in Table 3 below.

TABLE 3 Composition of diets fed to the different groups from d22 to d57Amount Amout Amount Amount in in in in diet of diet of diet of diet ofControl 2FL LNnT HMO Mix Group Group Group Group [%] [%] [%] [%]Cornstarch 53.4 53.4 53.4 53.4 Casein 20 20 20 20 Sucrose 10 10 10 10Soybean oil 7 7 7 7 Mineral Mix AIN-93-G * 3.5 3.5 3.5 3.5 Cholinebitartrate 0.25 0.25 0.25 0.25 L-Cystine 0.3 0.3 0.3 0.3Tert-butylhydroquinone 0.0014 0.0014 0.0014 0.0014 Vitamin MixAIN-93-VX * 1 1 1 1 Maltodextrin 4.5 0 0 0 2FL 0 4.5 0 3 LNnT 0 0 4.51.5 * from Research Diets, Inc

Rats were sacrificed at day 57 after a 6-hr fasting period and samplesof the small intestine were collected. The thickness of the muscle layerwas measured on a sample of the ileum.

One 1-cm sample from the ileum (1 cm cranial from caecum) was collectedand flushed with PBS (Sigma D8537). This section was fixed and processedaccordingly for histological and morphometric analysis.

Two consecutive 4 μm-thick sections of fixed tissue were stainedmanually with hematoxylin and eosin to determine muscle layer thickness.Muscle layer thickness was captured by light microscopy using a dry 10×objective, using a computerized image analyser system Axion Vision(version 4.8.2.0, Zeiss®).

Findings

The thickness of the intestinal layer measured in each rat isrepresented in FIG. 1. For each group, the mean muscle thickness isrepresented by a horizontal line. As can be seen, the mean musclethickness of the Normal group and of the IUGR group were similar,indicating that the growth retardation did not have significant impacton the muscle thickness. An increase of the muscle thickness wasobserved in the IUGR/LNnT group showing the beneficial effect of thishuman milk oligosaccharide on development of the intestinal muscle. Incontrast the IUGR/2FL group exhibited no increase in intestinal musclethickness. The beneficial effect of LNnT was confirmed even when LNnTwas used in smaller amount (three times lower amount), in combinationwith 2-FL, as evidenced by the intestinal muscle thickness measured inthe IUGR/HMO Mix, which was not significantly different from thatmeasured in the IUGR/LNnT group. This latter result indicates that thepositive effect of LNnT is maintained at lower dosage and that itspositive effect is not negatively impacted by the presence of otherHMOs, like 2-FL.

These results show that LNnT alone or in combination with 2-FL wassuccessful in increasing the thickness and thus the mass, of the musclein the ileum of the young rats. Such human milk oligosaccharide thuspromoted the development and growth of the intestinal muscle in theileum. Such increase in muscle thickness is associated with promotion ofthe contractile capacity of the intestine, leading to improvedgastrointestinal motility.

Example 4

A supplement for preterm infants is prepared, such as to provide thefollowing daily dose:

-   -   0.034 g/kg of body weight per day of LNnT; and    -   0.34 g/kg of body weight per day of 2-FL

1. A method of promoting the intestinal motility in an infant or a youngchild comprising administering a nutritional composition comprising atleast one N-acetylated oligosaccharide to a child in need of same.
 2. Amethod according to claim 1, wherein the method of promoting theintestinal motility is a method of promoting the motility in the smallintestine.
 3. A method according to claim 1, for use in a method ofpromoting the intestinal contractile capacity in an infant or a youngchild.
 4. A method according to claim 3, wherein the method of promotingthe intestinal contractile capacity is a method of promoting thecontractile capacity in the small intestine.
 5. A method according toclaim 1, for use in a method of promoting the development and/or thegrowth of intestinal muscles in an infant or a young child, respectivelyin a child.
 6. A method according to claim 5, wherein the method ofpromoting the development and/or the growth of intestinal muscles is amethod of promoting the development and/or the growth of the muscles ofthe small intestine.
 7. A method according to claim 1, for use in amethod of increasing the intestinal muscle thickness in an infant or ayoung child.
 8. A method according to claim 7, wherein the method ofincreasing the intestinal muscle thickness is a method of increasing thethickness of the muscle in the small intestine.
 9. A method according toclaim 1, for use in a method of promoting the enteral feeding tolerancein an infant or a young child.
 10. A method according to claim 1, foruse in a method of preventing and/or treating small intestinal bacterialovergrowth (SIBO).
 11. A method according to claim 1, wherein theN-acetylated oligosaccharide is lacto-N-neotetraose (LNnT),lacto-N-tetraose (LNT), para-lacto-N-neohexaose (para-LNnH),disialyllacto-N-tetraose (DSLNT) or any combination thereof.
 12. Amethod according to claim 11, wherein the N-acetylated oligosaccharideis lacto-N-neotetraose (LNnT).
 13. A method according to claim 1,wherein the composition comprises at least one additionaloligosaccharide.
 14. A method according to claim 13, wherein thecomposition comprises at least one fucosylated oligosaccharide.
 15. Amethod according to claim 1, wherein the N-acetylated oligosaccharide(s)is/are in a total amount of 0.005-3 g/L of the composition and/or in atotal amount of 0.004-2.3 g/100 g of composition on a dry weight basis.16-17. (canceled)
 18. A method according to claim 1, wherein theN-acetylated oligosaccharide(s) is/are in a total amount of 0.09-0.8 g/Lof the composition and/or in a total amount of 0.07-0.6 g/100 g ofcomposition on a dry weight basis.
 19. A method according to claim 1,wherein the composition comprises at least another oligosaccharide(s)and/or fiber(s) and/or precursor(s) thereof selected from the groupconsisting of galacto-oligosaccharides (GOS), fructo-oligosaccharides(FOS), xylooligosaccharides (XOS), inulin, polydextrose, sialylatedoligosaccharides, sialic acid, fucose and any combination thereof.
 20. Amethod according to claim 1, wherein the composition comprises at leastone probiotic in an amount of from 10³ to 10¹² cfu/g of the composition(dry weight).
 21. A method according to claim 1, wherein the nutritionalcomposition is an infant formula, a starter infant formula, a follow-onor follow-up infant formula, a growing-up milk, a baby food, an infantcereal composition, a fortifier or a supplement.
 22. A method accordingto claim 1, wherein the infant is a preterm infant, an infant having alow birth weight, an infant born small for gestational age (SGA) and/ora growth-retarded infant or young child.
 23. (canceled)